PROJECT SUMMARY. Metazoan mitochondria contain tRNAs which are highly diverged in both sequence and structure from those found elsewhere in evolution. These tRNAs are recognized and specifically charged with their cognate amino acids by nuclearly encoded mitochondrial aminoacyl-tRNA synthetases. Although the enzymes have a high degree of sequence similarity to cytoplasmic and bacterial counterparts, examination of their unusual tRNA substrates suggests that novel modes of specific RNA recognition may be employed. Metazoan mitochondrial alanyl-tRNA sythetases (AlaRSs) are of special interest because the tRNA acceptor stem sequence that defines alanine identity in all other systems is extremely variable in animal mitochondria. The objective of the research proposed here is to elucidate the specific adaptations of human mitochondrial AlaRS that allow this enzyme to recognize its bizarre tRNA substrate. Our preliminary results show RNA recognition properties of previously characterized homologues from bacteria, eukaryotes and other metazoan mitochondria poorly predict those of the human mitochondrial enzyme. The basis of substrate specificity in the human mitochondrial system will be determined by mapping tRNA identity elements using three approaches: construction of hybrid tRNAs, cross-species aminoacylation of other animal mitochondrial tRNAs, and nucleotide analog interference mapping. These experiments will guide the preparation of mutants to assess the importance of individual nucleotides. The research will also determine the adaptations in the mitochondrial enzyme responsible for altered specificity using deletion analysis to establish which of the conserved RNA binding domains of the protein are involved in specific recognition. Site-directed mutation and domain swaps will be used to examine the roles of specific side chains or peptides. RELEVANCE. Because aminoacyl-tRNA synthetases are an essential family of enzymes with cross- species differences in specificity, they make attractive targets for antibiotic development. Defects in aaRS recognition of tRNA are also involved in several inheritible mitochondrial diseases. This work will extend our basic understanding of the evolution and specificity of this important enzyme class, an understanding that is vital to the continuing development of health-related applications.